Recombinant DNA Flashcards
(17 cards)
Definition of Recombinant DNA.
Combining DNA molecules from different sources to create the recombinant DNA by using artificial means.
2 principles of recombinant DNA.
(1) DNA in all organisms has the same molecular structure.
(2) genetic codes are universal to all organisms.
outline the steps of recombinant DNA technology.
(1) isolate target gene from donor cell
(2) cutting of vector
(3) ligation of target gene and vector
(4) replication of host cell
(5) gene product
What enzymes are used in Recombinant DNA tech.
(1) Restriction enzymes
(2) DNA ligase
What is the function of restriction enzymes ?
(1) catalyse the breakdown of linkage between nucleotides in DNA.
(2) cuts DNA at specific base sequence.
(3) act as a molecular scissor.
What is the function of DNA ligase ?
(1) catalyse the joining of nucleotides together.
(2) act as molecular glue.
What is commonly used as a vector in recombinant DNA ?
Bacterial Plasmid
Structure of a plasmid
(1) Small
(2) Circular
(3) double-stranded piece of DNA
Adaptations of plasmid.
(1) small for easy handling
(2) replicate independently
(3) can be transferred naturally by transformation
(4) usually carry resistance gene: can survive in different chemicals
(5) can be easily transformed into bacteria for producing gene product
What is the use of screening ?
For selecting bacterial cells that have taken up the recombinant plasmid.
What can be a selectable marker ?
antibiotic resistance gene.
Give an example of selectable marker.
Amp^R gene.
An ampicillin resistance gene.
Give two possible reasons for failure in gene expression.
(1) fragment digested from the vector was ligated back to the vector instead of the insert.
(2) the insert enters an inappropriate site since the restriction enzymes may cuts open an incorrect position.
Can the bacteria that received plasmid that failed gene expression go through selection ?
Yes.
As long as they have antibiotic resistance gene.
How can a target gene be extracted ?
By adding restriction enzymes to cut the target gene from DNA fragment of the donor cell at restriction site.
Feature of cut ends of target gene.
(1) sticky end
(2) single stranded with unpaired bases
What should be done to the extracted target gene ?
Amplify the gene by PCR
